Some biological cells, either by natural mutation or through genetic engineering, lack specific enzymes required for either synthesizing an essential molecule or breaking down a toxic molecule. These cells, known as auxotrophs or auxotrophic organisms, are generally cultured with the nutrients that they require for survival. They can also be enabled to survive by providing either the deficient enzyme itself or a copy of the DNA coding for the deficient enzyme to the intracellular domain of the cell. A cell having all of the required metabolic machinery for survival is known as a prototroph, or a prototrophic organism.
For the purposes of this disclosure, the terms “auxotrophic” and “auxotroph” are used to signify living cells that are lacking one or more particular enzymes in a metabolic pathway critical for survival of the cell in the desired environmental conditions. The terms “prototrophic” and “prototroph” are used to signify living cells capable of survival in the desired environmental conditions without modification. Thus, “prototroph” is an antonym of “auxotroph.”
In some applications using genetically modified cells or organisms, it is important to limit the escape potential of the cell or organism. Often auxotrophic systems are used for such applications. For example, the use of genetically modified organisms in field applications requires mechanisms that are designed to limit the escape potential of the genetically modified cells or their modified DNA into the gene pool of the natural ecosystem. One mechanism for limiting the escape potential of auxotrophic cells to the surrounding ecosystem is to provide an essential nutrient for use by the cells that is not found in the local environment. This allows a zone of survival for the auxotrophic cells around the point of administration of the nutrient and limits the growth of the cells outside the desired region. Often time-release nutrient matrices are used within the desired region of survival.
However, there are some problems inherent in the foregoing approaches. For example, the depletion of the nutrient will eventually cause the cells to die, the local environment may be contaminated by leaching of the nutrient or its byproducts, or a nutrient gradient may be formed that forces a selective pressure on the cells leading to the evolutionary transformation of the cells from auxotrophs to prototrophs, allowing their escape into the environment.
Another method of containment for auxotrophic cells physically contains them within a barrier, such as a membrane or matrix having pore sizes smaller than the cells. Limitations of this approach include the reduced ability of material to flow to and from the cells due to physical properties of the barrier, and the potential of the barrier to breach and release the cells contained within.
Despite the foregoing, there continues to be a need for improved methods for limiting the escape potential of cells and that can provide a high level of survival for the cells within a limited area and also reduce the environmental impact of maintaining the organism, as well as reduce the escape potential of the organism. The introduction of an exogenous substrate immobilized compartmentalized component to the cell is therefore a preferred embodiment of the present invention that ensures cellular survival and minimal environmental impact, yet prevents transfer of the component from the compartment by restricting the component's ability to interact with cellular chromosomes and other large cellular macromolecules. Thus the cell is transiently transformed from an auxotroph to a substrate-dependent prototroph. Growth off the substrate will result in cell death, and daughter cells are not able to inherit the component due to the compartmentalization.
In accordance with a first aspect, one exemplary embodiment of the disclosure provides a method for the transient transformation of at least one living biological cell having an intact cell membrane defining an intracellular domain. The method includes introducing at least one compartmentalized exogenous component to the intracellular domain of the cell, wherein the component is fixed within a recessed compartment at a tip of a cellular penetrant structure being dimensioned to extend through the cell membrane into the intracellular domain without significantly damaging the cell, and wherein the cell is penetrated by at least a portion of the tip of the penetrant, and wherein the component is retained within the compartment and wherein the compartment is dimensioned to restrict interaction of the component with cellular macromolecules.
In accordance with a second aspect, the disclosure provides an apparatus for the transient transformation of at least one living biological cell having an intact cell membrane defining an intracellular domain. The apparatus includes an immobilized cellular penetrant structure having a tip with a recessed compartment dimensioned to extend through the cell membrane without significantly damaging the cell, and at least one extracellular component fixedly attached to at least a portion of the surface of the recessed compartment.
An advantage of the disclosed embodiments is the transient introduction of a compartmentalized exogenous component fixedly attached within the tip of a cellular penetrant structure to the intracellular domain of the cell. The component transforms the cell, but only while the penetrant structure remains within the dependent cell. Cell growth off the penetrant structure would result in cell death. The immobilization of the essential component within a compartment enables functional activity of the component within the cell but does not allow the component to be passed from one cell to another or to be inherited by daughter cells. The compartmentalization of this component prevents the transfer of the component from the compartment and into the host cell by restricting the access of the component to other large cellular macromolecules such as chromatin or replication holoenzymes. The compartmentalization can also act to protect the component from degradation by cellular enzymes, thus providing for longer activity of the delivered component.
The exogenous component may be a gene or set of genes that encode a specific enzyme or set of enzymes required by the cell either to synthesize a required nutrient or to degrade a toxic agent. The component may be the required enzyme or set of enzymes itself, fixedly attached in a manner that allows catalytic functionality within the cell. The component may also be another molecule or set of molecules that influences or assists in the required reactions within the cell. The component may also be a combination of at least one gene and at least one enzyme required for posttranscriptional modification of the gene message, each subcomponent attached fixedly to a surface of the recessed compartment of the cellular penetrant substrate.
A further advantage of the disclosed embodiments is due to the attachment of the enzyme, DNA, or nutrient component within a recessed compartment at the tip of an immobile cellular penetrant structure on a substrate. By such attachment the cells or organisms can be forced to reside only on the substrate while being mounted on the penetrant structure and the component is prevented from any inheritable incorporation into the cell. Immobilization of the cells on the cellular penetrant substrate limits the potential of the cells to escape from the substrate.